The long term objective of this research is to identify both the viral and cellular factors which contribute to the diversity in host range, virulence, and antigenic properties exhibited by the influenza A viruses. Identifying these factors is key to understanding the strategies by which these viruses survive in nature and continue to produce pandemic diseases. This proposal focusses on the role of the hemagglutinin (HA) in this process and is designed to identify both alterations in the HA gene and host-determined cellular processes that influence the receptor binding properties of this virion glycoprotein. The effects of HA sequence variation in receptor binding and host range at the cellular level will be investigated by characterizing a collection of HA variants which differ from each other by a single amino acid in the HA1 subunit. Variants already isolated and those to be generated by site specific mutagenesis will be used to evaluate the roles of specific amino acids and specific glycosylation sites in determining the function and stability of the HA. The properties of these altered HAs will he examined by expressing them in eukaryotic cells and by incorporating altered HA genes into virions. The role of oligosaccharide structure in determining the receptor binding and host range properties will be evaluated by characterizing the oligosaccharides put onto the HA at specific sites by different host cells and by altering the HA oligosaccharides using inhibitors of glycoprotein processing. Neoglycolipids and neoglycoproteins, each containing a single kind of sialylated oligosaccharide, will be used to determine the receptor binding properties of the variants. These properties will be related to the ability of the variants to grow in cultured cells of different species. The mechanisms by which receptor binding is altered by genetic change and host cell modification will be determined. Since virus neutralization by antibodies is determined in part by the affinity of the HA for the receptors on host cells, the information obtained will he relevant to vaccine design and may provide clues for the prevention and control of future pandemics resulting from interspecies transfer of influenza viruses. The information will also be applicable to the search for antiviral agents. Lastly, the procedures developed for investigating the initial steps of viral infection and host range should prove invaluable for studying other viruses, especially those like human immunodeficiency virus which interact with cellular receptors via a glycoprotein.